Differential involvement of phosphoinositide 3-kinase/Akt in human RPE MCP-1 and IL-8 expression

Invest Ophthalmol Vis Sci. 2004 Jun;45(6):1887-96. doi: 10.1167/iovs.03-0608.

Abstract

Purpose: To investigate the role of the phosphatidylinositol 3-kinase (PI3K) pathway and the signal mediator AP-1 in monocyte chemotactic protein (MCP)-1 and interleukin (IL)-8 gene expression in human retinal pigment epithelial (hRPE) cells.

Methods: hRPE cells were stimulated with IL-1beta and TNF-alpha and by coculturing with monocytes in the presence or absence of a series of kinase inhibitors. The induction of MCP-1 and IL-8 protein and mRNA was determined by ELISA and RT-PCR, respectively. Western blot analysis, kinase assays, and electrophoretic mobility shift assays were used to detect the activation of signaling mediators and transcription factors.

Results: Concomitant with the induction of chemokine expression by the stimuli, there was phosphorylation of PI3K and its downstream targets-namely, Akt, GSK, and FKHR. Ly294002, a specific inhibitor of PI3K, resulted in time- and dose-dependent blockade of MCP-1 mRNA expression and protein production. The IC(50) for inhibition of MCP-1 secretion induced by IL-1beta, TNF-alpha, and hRPE-monocyte binding was 16, 12, and less than 3 micro M, respectively. In contrast, Ly294002 did not inhibit the IL-8 expression induced by any of the stimuli. Ly294002 as well as U0126, SB202190, and SP600125, the selective inhibitors of MEK, p38, and JNK, respectively, strongly inhibited induced c-fos expression, whereas Ly294002 did not inhibit induction of MEK, p38, or JNK. Blockade of PI3K/Akt abolished IL-1beta-induced nuclear translocation of AP-1, whereas the induction of IkappaB degradation was unchanged.

Conclusions: The Ly294002-sensitive PI3K/Akt pathway regulates MCP-1, but not IL-8 expression in hRPE cells independent of MAPK and IkappaB. PI3K-dependent induction of hRPE c-fos and AP-1 nuclear translocation may be a target for therapies aimed at modulating MCP-1 in retinal diseases.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Blotting, Western
  • Cells, Cultured
  • Chemokine CCL2 / genetics*
  • Chemokine CCL2 / metabolism
  • Coculture Techniques
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / pharmacology
  • Enzyme-Linked Immunosorbent Assay
  • Gene Expression Regulation / physiology*
  • Humans
  • Interleukin-1 / pharmacology
  • Interleukin-8 / genetics*
  • Interleukin-8 / metabolism
  • Monocytes / physiology
  • Phosphatidylinositol 3-Kinases / physiology*
  • Phosphoinositide-3 Kinase Inhibitors
  • Phosphorylation
  • Pigment Epithelium of Eye / drug effects
  • Pigment Epithelium of Eye / metabolism*
  • Protein Serine-Threonine Kinases*
  • Proto-Oncogene Proteins / antagonists & inhibitors
  • Proto-Oncogene Proteins / physiology*
  • Proto-Oncogene Proteins c-akt
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction
  • Time Factors
  • Transcription Factor AP-1 / metabolism
  • Tumor Necrosis Factor-alpha / pharmacology

Substances

  • Chemokine CCL2
  • Enzyme Inhibitors
  • Interleukin-1
  • Interleukin-8
  • Phosphoinositide-3 Kinase Inhibitors
  • Proto-Oncogene Proteins
  • RNA, Messenger
  • Transcription Factor AP-1
  • Tumor Necrosis Factor-alpha
  • AKT1 protein, human
  • Protein Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt